Le Centre de recherche Douglas

Single-cell and single-nucleus sequencing techniques are a burgeoning field with various biological, biomedical and clinical applications. Numerous high- and low-throughput methods have been developed for sequencing the RNA and DNA content of single cells. However, for all these methods, the key requirement is high-quality input of a single-cell or single-nucleus suspension. Preparing such a suspension is the limiting step when working with fragile, archived tissues of variable quality. This hurdle can prevent such tissues from being extensively investigated with single-cell technologies. We describe a protocol for preparing single-nucleus suspensions within the span of a few hours that reliably works for multiple postmortem and archived tissue types using standard laboratory equipment. The stages of the protocol include tissue preparation and dissociation, nuclei extraction, and nuclei concentration assessment and capture. The protocol is comparable to other published protocols but does not require fluorescence-assisted nuclei sorting (FANS) or ultracentrifugation. The protocol can be carried out by a competent graduate student familiar with basic laboratory techniques and equipment. Moreover, these preparations are compatible with single-nucleus (sn)RNA-seq and assay for transposase-accessible chromatin (ATAC)-seq using the 10X Genomics Chromium system. The protocol reliably results in efficient capture of single nuclei for high-quality snRNA-seq libraries.